RS61954B1 - Novel probiotic bacterial strain of lactobacillus plantarum and compositions and uses thereof in the treatment of inflammation - Google Patents

Description

Description

[0001] The present invention relates to a new strain of the bacterium Lactobacillus plantarum Gos 42 (DSM 32131) and its compositions for use as a probiotic. The new strain has a special application in medicine, especially in the treatment and/or prevention of inflammation. The new strain and its compositions find particular application in the treatment and/or prevention of inflammation in the oral cavity, preferably for the treatment and/or prevention of gingivitis and/or periodontitis.

[0002] In particular, the new strain and its compositions can be used as anti-inflammatory agents to reduce or inhibit the release of one or more inflammatory factors belonging to the group that includes interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), isoprostane, matrix metallopeptidase 9 (MMP9) and NF-kB.

[0003] Preferably, the new strain of the invention is a probiotic, that is, a microorganism that is useful when grown in a certain microenvironment, e.g. by inhibiting or preventing the growth of other organisms in the same microenvironment. Examples of probiotic microorganisms include lactobacilli, which can colonize the gastrointestinal tract, at least temporarily displace or destroy pathogenic organisms, as well as be of other benefit to the host.

[0004] Here, "probiotic" refers to microorganisms that form at least part of the transient or endogenous flora and on that occasion show a favorable prophylactic and/or therapeutic effect on the host organism. Probiotics are generally known to be clinically safe (eg, non-pathogenic) by those skilled in the art. By way of example, and not by way of limitation to any particular mechanism, the prophylactic and/or therapeutic effect of the acid-producing bacteria of the present invention is obtained, in part, by competitive inhibition of pathogen growth due to: (i) their superior colonization abilities; (ii) parasitism of unwanted microorganisms; (iii) acid production (eg lactic, acetic and other acidic compounds) and/or other extracellular products that have antimicrobial activity; and (iv) its various combinations. It should be noted that the said products and activities of the acid-producing bacteria of the present invention work synergistically to produce the beneficial probiotic effect described herein.

[0005] By a purified and isolated preparation of a bacterial strain, it is meant that the preparation does not contain another species or strain of bacteria in an amount that is sufficient to affect the replication of the preparation at a certain temperature. The purified or isolated preparation of the bacterial strain is performed using standard methods, e.g. plating at limited dilution and temperature selection.

[0006] Inflammatory conditions of the gums are primarily caused by the formation of dental plaque. Colonies of bacteria form a biofilm on the tooth surface due to the presence of food residues as well as salivary components. If it is not removed sufficiently in the early stage, the plaque coating on the surface of the teeth leads to the deposition of tartar, which is very difficult to remove. The presence of an increased number of bacteria on the gingival margin leads to inflammation of the gingiva, known as gingivitis. In susceptible individuals, gingivitis can lead to periodontitis, which can lead to tooth loss. Especially lipopolysaccharides (LPS) present in Gram-negative bacteria can cause a non-specific immune response in LPS-stimulated macrophages, which release prostaglandin E2 (PEG2) and pro-inflammatory mediators such as interleukins and TNF-α in the affected tissue. Proinflammatory mediators induce the release of additional PGE2 and matrix metalloproteinases (MMPs) from existing fibroblasts, which destroy the extracellular matrix of the surrounding tissue. This allows bacteria to penetrate deeper into the tissue and promote the inflammatory process independent of the outer layer of the epithelium and dental root, causing the formation of a periodontal pocket. The alveolar bone, which holds the tooth in place, resorbs in the face of advancing bacteria and causes the tooth to become unstable and fall out if left untreated.

[0007] In order to avoid the progressive destruction of the gums, inflammatory responses in the oral cavity must be suppressed at an early stage, and it is best to prevent them.

[0008] Many different approaches have addressed this problem, from advanced methods for mechanical plaque removal to oral care products with strong antibacterial properties.

[0009] Admittedly, not all bacteria present in the oral cavity are associated with diseases, and many even promote oral health. Therefore, it is preferable to establish a balance of the healthy composition of the oral microbiota instead of non-specific eradication of the bacteria present.

[0010] The normal oral microbiota is very complex and includes about 700 species of bacteria as well as archaea, fungi, protozoa and viruses. Lower gut commensals such as lactobacilli and bifidobacteria have been shown to have beneficial effects on gut health, including some anti-inflammatory properties, when administered as probiotics.

[0011] The probiotic effect of bacteria in the oral cavity has been the subject of some research, but the results vary greatly in relation to the species used, and the appropriate parameters for effective application are difficult to determine because the effect can largely rely on unrelated effects.

[0012] Of the probiotic effect, the general antibacterial properties against the species associated with the disease, the reduction or prevention of bacterial adhesion to the tooth surface, and the anti-inflammatory effects were discussed in the literature.

[0013] EP 2420580 A1 relates to a composition containing an effective amount of Lactobacillus plantarum strain CECT 7481 and Lactobacillus brevis strain CECT 7480, the use of said composition as a probiotic and/or as a medicine for oral administration as well as products containing said composition.

[0014] JP 2014-000039 A relates to lactobacillus for the prevention of caries, prevention/treatment of periodontal disease, improvement/prevention of bad breath, and suppression of biofilm formation in the oral cavity at normal times (at the time of non-eating), or the culture and culture sediment of this lactobacillus, its neutralized products, the composition containing it, and the like. Lactobacillus is selected from the group of strains Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus sakei, Enterococcus avium, and Enterococcus durans

[0015] WO 2014/140080 A1 relates to bacterial preparations of Lactobacillus plantarum NCC 2936 (CNCM I-4026) and to a composition for use in improving oral health, containing the bacterial preparation of Lactobacillus plantarum NCC 2936.

[0016] US 2014/023620 A1 relates to probiotic compositions containing a mixture of at least two probiotic bacteria, including Streptococcus salivarius K12® (BLIS K12®), such as Streptococcus salivarius K12® (BLIS K12®) BAA1024, and at least one Lactobacillus bacterium, such as one comprising those known to be suitable for oral administration, e.g. of those known to improve the recipient's health, particularly the recipient's oral health.

[0017] WO 2010/064373 A1 relates to an IL-8 inhibitor for inhibiting the production of IL-8, which contains a culture pellet of a lactic acid bacterium obtained from plants carrying a 16SrRNA gene containing the nucleotide sequence shown in any of SEQ ID NO:1 to SEQ ID NO:4 or an extract from the culture pellet.

[0018] DE 202009011379 U1 refers to pastes containing probiotics and preparations for oral care, which contain probiotic cultures from the group that includes bacilli, actinobacteria, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus para casej, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salvarius, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Leuc. Mesenteroides, Ped. Acidilactici, Sporolactobacillus inulinus, Streptococcus thermophilus, Bacilus cereus, Escherichia coli, Propionibacterium freudenreichii and Saccharomyces cerevisiae.

[0019] However, little is known about the influence of specific strains of probiotics on inflammatory mechanisms, especially regarding the suppression of various inflammatory mediators. Exceptionally, during the extensive research that led to the subject invention, it was discovered that some strains of generally accepted probiotic bacteria can enhance the release of pro-inflammatory factors in certain doses. Therefore, there remains an unmet need for new strains, which show a desirable effect on pro-inflammatory and anti-inflammatory mediators of inflammation.

[0020] The task of this invention is to provide a new bacterial strain and its composition, which can be used in medicine, especially in the treatment and/or prevention of inflammation, such as occur in the oral cavity, especially in the treatment and/or prevention of gingivitis and/or periodontitis.

[0021] An additional task of the present invention is to provide a new bacterial strain and its compositions, which have the ability to reduce or inhibit the release of one or more inflammatory factors such as interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), isoprostane, matrix metallopeptidase 9 (MMP9) and NF-kB.

[0022] The task of the present invention is fulfilled by providing the strain GOS 42 of Lactobacillus plantarum, and its compositions, deposited by Probi AB of Sölvegatan 41, 22370 Lund, Sweden at the Leibnitz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7 B, D-38124 Braunschweig in accordance with the requirements of the Budapest Agreement dated September 2, 2015 and received the deposit accession number DSM 32131.

[0023] The strain of the invention was isolated from the saliva of healthy human volunteers and selected from among 50 candidate probiotic strains including strains of Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifdobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus LAFTI, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus salivarius, Streptococcus thermophilus and Lactococcus lactis.

[0024] Through extensive screenings, the bacterial strain according to the invention showed a different modulating activity, mainly by inhibiting the release of certain pro-inflammatory factors while, at the same time, not releasing, resp. slightly releases other pro-inflammatory factors. The present invention now enables the optimized use of a probiotic bacterial strain in the prevention and/or treatment of inflammatory conditions, particularly those occurring in the oral cavity, which was not previously possible.

[0025] As explained above, the colonization of the oral mucosa by bacteria associated with diseases and the formation of plaque can direct the microbiological balance in the oral cavity towards the accumulation of harmful microorganisms, which is also called dysbiosis. Therefore, the use of microorganisms in the prevention and/or treatment of inflammation in the oral cavity according to the invention includes the use in the prevention and/or treatment of plaque or plaque-induced diseases and, as an advantage, helps to avoid oral dysbiosis by balancing the oral flora so as to lead to a healthy state.

[0026] In a preferred embodiment of the present invention, the microorganisms described above are attenuated or dead, preferably inactivated by heat, ideally by incubation for 2 to 8 minutes at a temperature between 70 and 100 °C.

[0027] In the studies described below, it has been shown that the microorganisms according to the invention can have an inhibitory effect on the release of proinflammatory factors even in an inactivated state. Therefore, it is also possible to use a new strain if it is dead or heat-inactivated. Exceptionally, heat-inactivated strains can show the same or even slightly improved anti-inflammatory activity according to certain factors.

[0028] In one aspect, the subject invention relates to the above-mentioned microorganisms for use as an anti-inflammatory agent, especially for reducing or inhibiting the release of one or more inflammatory factors selected from the group that includes interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), isoprostane, matrix metallopeptidase 9 (MMP9) and NF-kB.

[0029] In accordance with an additional aspect of the invention, fragments of a new strain of microorganism as defined in all aspects described above, can be used in the treatment and/or prevention of inflammation, especially in the oral cavity, and preferably in the treatment and/or prevention of gingivitis and/or periodontitis.

[0030] It is not necessary to use whole cells of the new strain of the invention since mixtures containing only fragments (eg, the remains of degraded cells) of the microorganism are sufficient to provide the inventive effect.

[0031] In an additional aspect, the present invention also relates to a pharmaceutical composition, which contains Lactobacillus plantarum GOS 42 or its fragments and a pharmaceutically acceptable carrier, excipient and/or diluent. The total amount of the microorganism or its fragments is preferably sufficient for the treatment and/or prevention of inflammation, especially in the oral cavity, preferably for the treatment and/or prevention of gingivitis and/or periodontitis. The total amount of the microorganism or its fragments is preferably in the range from 0.01 to 100%, preferably in the range from 0.1 to 50%, most preferably from 1 to 10%, in each case in relation to the total weight of the composition and/or where the total amount of the microorganism(s) or its/their fragment(s) is in the range of 1 x 10<3> to 1 x 10<11> units colony-forming units (CFU), preferably in the range of 1 x 10<5> to 1 x 10<10>SFU, and ideally from 1x10<8> to 1 x 10<9>CFU.

[0032] In a preferred embodiment, the microorganisms (bacteria) of the invention are present in the composition at a concentration of approximately 1 x 10<3> to 1 x 10<14> per gram of colony forming units (CFU). Preferably about 1 x 10<5>to 1 x 10<12>CFU/gram, with other embodiments having concentrations of about 1 x 10<9>to 1 x 10<13>CFU/gram, about 1 x 10<5>to 1 x 10<7>CFU/gram, or about 1 x 10<8>to 1 x 10<9>CFU/gram. However, it will be appreciated that it is the total CFU rather than the concentration that is most important.

[0033] In one form of carrying out the bacteria of the invention is in a pharmaceutically acceptable carrier or food suitable for oral administration to a mammal, preferably as a powdered nutritional supplement, a pellet formulation or a liquid formulation. The mammal is preferably a human.

[0034] The expert in the field is aware that the probiotic organism used in the composition or product according to the invention is a biological material whose activity can vary depending on the batch and also depends on the production and processing procedure. Therefore, the appropriate amount can be adjusted according to the given range.

[0035] Furthermore, the present invention relates to a composition or product as described above for use in medicine, preferably in the treatment and/or prevention of inflammation, especially in the oral cavity, and most preferably for the treatment and/or prevention of gingivitis and/or periodontitis.

[0036] The composition according to the invention may further contain one or more components selected from the group consisting of carriers, excipients or additional active ingredients such as, for example, active agents from the group of non-steroidal anti-inflammatory drugs, antibiotics, steroids, anti-TNF-alpha antibodies or other biotechnologically produced active agents and/or substances as well as analgesics, dexpanthenol, prednisolone, polyvidone iodide, chlorhexidine-bid-D-gluconate, hexetidine, benzydamine HCL, lidocaine, benzocaine, macrogol lauryl ether, benzocaine in combination with cetidyl pyridinium chloride or macrogol lauryl ether in combination with protein-free hemodialysate from calf blood, as well as for example fillers (e.g. cellulose, calcium carbonate), plasticizers or flow improvers (e.g. talc, magnesium stearate), coatings (e.g. polyvinyl acetate phthalate, hydroxyl propyl methyl cellulose phthalate, disintegrants (eg starch, cross-linked polyvinyl pyrrolidone), softener (e.g. triethyl citrate, dibutyl phthalate), granulating substances (lactose, gelatin), retardation (e.g. poly (meth) acrylic acid methyl/ethyl/2-trimethyl aminomethyl ester copolymers in dispersion, vinyl acetate/crotonic acid copolymers), compacting (e.g. microcrystalline cellulose, lactose), solvents, suspending or dispersing agents (e.g. water, ethanol), emulsifiers (e.g. cetyl alcohol, lecithin), substances for modifying rheological properties (silica, sodium alginate), substances for microbial stabilization (e.g. benzalkonium chloride, potassium sorbate), preservatives and antioxidants (e.g. DL-alpha-tocopherol, ascorbic acid), substances for modifying pH (lactic acid, citric acid), foaming agents or inert gases (e.g. fluorinated chlorinated hydrocarbons, carbon dioxide), colors (iron oxide, titanium oxide), basic ingredients for pomades (e.g. paraffins, beeswax) and others as described in the literature (e.g. in Schmidt, Christin. Wirk- und Hilfsstoffe für Rezeptur, Defektur und Großherstellung. 1999; Wissenschaftliche Verlagsgesellschaft mbH Stuttgart or Bauer, Frömming Führer. Lehrbuch der Pharmazeutischen Technologie. 8th edition, 2006. Wissenschaftliche Verlagsgesellschaft mbH Stuttgart).

[0037] The composition or product according to the present invention may also be coated or encapsulated.

[0038] Encapsulation of the composition according to the invention may have the advantage of allowing controlled release, for example upon contact with water, or continuous release over a long period of time. Moreover, the composition can be protected from degradation, which improves the shelf life of the product. Methods of encapsulation of active ingredients are well known in the art and numerous encapsulation materials are available, as well as methods of their application to the composition in accordance with special requirements.

Furthermore, the composition or product according to the invention can be in the form of a solution, suspension, emulsion, tablet, granule, powder or capsule.

[0040] The composition or product according to the invention can be selected from the group comprising toothpaste, tooth gel, tooth powder, dental cleaning liquid, dental cleaning foam, rinsing liquid, mouth spray, dental floss, chewing gums and lozenges.

[0041] Such compositions or products may contain abrasive systems (abrasive components and/or polishing components) such as silicates, calcium carbonate, calcium phosphate, aluminum oxide and/or hydroxyl apatite, surfactants such as e.g. sodium lauryl sulfate, sodium lauryl sarcosinate and/or cocamidopropyl betaine, humectants such as glycerol and/or sorbitol, thickening agents, e.g. carboxymethyl cellulose, polyethylene glycols, carrageenans and/or Laponite®, sweeteners such as saccharin, flavorings and flavor enhancers due to unpleasant taste sensation, taste modifying substances (e.g. inositol phosphate, nucleotides, e.g. guanosine monophosphate, adenosine monophosphate or other substances, e.g. sodium glutamate or 2-phenoxypropionic acid), cooling agents such as menthol derivatives (e.g. L-menthyl lactate, L-menthyl alkyl carbonate, menthone ketals), icilin and icilin derivatives, stabilizers and active agents such as sodium fluoride, sodium monofluorophosphate, tin difluoride, quaternary ammonia fluoride, zinc citrate, zinc sulfate, tin pyrophosphate, tin difluoride, mixtures of various pyrophosphates, triclosan, cetyl pyridinium chloride, aluminum lactate, potassium citrate, potassium nitrate, potassium chloride, strontium chloride, hydrogen peroxide, flavors, sodium bicarbonate and/or agents for correction.

[0042] Chewing gum or dental care chewing gum may contain a chewing gum base containing elastomers e.g. polyvinyl acetate (PVA), polyethylene, (low or medium molecular), polyisobutane (PIB), polybutadiene, isobutene/isoprene copolymers, polyvinyl ethyl ether (PVE), polyvinyl butyl ether, vinyl ester and vinyl ether copolymers, styrene/butadiene copolymers (SBR) or vinyl elastomers, e.g. based on vinyl acetate/vinyl laureate, vinyl acetate/vinyl stearate or ethylene/vinyl acetate in mixtures of said elastomers as for example described in EP 0242325, US 4,518,615, US 5,093,136, US 5,266,336 US 5,601,858 or US 6,986,709. Additionally, chewing gum bases may contain additional ingredients, e.g. (mineral) fillers, e.g. calcium carbonate, titanium dioxide, silicon dioxide, talc, aluminum oxide, dicalcium phosphate, tricalcium phosphate, magnesium hydroxide and its mixtures, plasticizers (e.g. lanolin, stearic acid, sodium stearate, ethyl acetate, diacetin (glycerol diacetate), triacetin (glycerol triacetate) and triethyl citrate), emulsifiers (e.g. phosphatides, such as lecithin and mono and diglycerides of fatty acids, e.g. glycerol monostearate), antioxidants, wax (eg paraffin wax, candelilla wax, carnauba wax, microcrystalline waxes and polyethylene waxes), fats and fatty oils (eg solid (hydrogenated) vegetable or animal fats) and mono, di or triglycerides.

Short description of the pictures:

Figure 1 shows the anti-inflammatory effects of Lactobacillus plantarum strain GOS 42 (DSM 32131) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), prostaglandin E2 (D), tumor necrosis factor alpha (E) and isoprostane (F). Left column refers to untreated cells, right column to dead cells. Figure 2 shows the anti-inflammatory effects of Lactobacillus plantarum GOS 42 (DSM 32131) in an attenuated form on interleukin in human gingival fibroblasts. The left column refers to interleukin 6, the right column to interleukin 8.

[0044] The following examples are added to illustrate the subject invention without limiting the scope.

Example 1: Cultivation and handling of probiotic strains

[0045] The strain of the invention is selected from over 50 candidate probiotic strains including strains of bacteria Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifdobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus LAFTI, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus salivarius, Streptococcus thermophilus and Lactococcus lactis.

[0046] In order to identify the optimal growth conditions and time of collection and to determine the colony forming units (CFU) for the screening of probiotic bacteria, the logarithmic phase at the end of the growth phase was first determined.

Bacterial growth

[0047] Frozen (-80°C) probiotic stocks were thawed overnight at 4°C, and 6 ml of sterile 9% NaCl solution was added to 1.2 ml of bacteria the following morning. The samples were centrifuged (5 min, 5000 oum), the precipitate was discarded, the pellets were washed with 8 ml of 9% NaCl and centrifuged again for 5 min at 5000 oum. The pellets were then resuspended in 1.2 ml of 9% NaCl and 1 ml of the sample was added to 50 ml of medium (MRS Bouillon, Carl Roth KG, Karlsruhe) at 37°C. Incubations were performed in a sterile polypropylene test tube (Greiner) of 50 ml, and samples were collected at different time intervals in order to evaluate the growth curve.

Determination of optical density

[0048] To determine the optical density, 500 µl of the bacterial suspension was removed and dissolved in 1 ml of MRS Bouillon in a PS cuvette (brand) of 1.5 ml. Determination of optical density was done at 600 nm ((ThermoScientific, Helios Epsilon) 1.5 ml of MRS Bouillon was used as a blank sample.

Determination of CFU

[0049] To determine CFU bacteria were dissolved (1:10,000,000, 1:50,000,000 and 1:100,000,000), placed on MRS agar media (MRS Agar, X924, Carl Roth) and incubated for 2 days at 37°C. Grown colonies were then counted and CFU was calculated.

[0050] The bacteria approached logarithmic phase from the start until 7 to 8 hours when they began to reach a plateau phase. The amount of bacteria seeded does not change the shape of the curve. 5 hours was chosen as the point of the steepest growth phase for the collection of bacteria in the logarithmic phase and 7 hours for their collection at the end of the logarithmic phase.

Example 2: Establishment of stimulation of human monocytes with probiotics

[0051] Stimulation of monocyte cell cultures with a new probiotic strain was established using strains obtained as a powder. First, several forms of application were tested, such as the use of cultured bacteria (collected in the logarithmic phase), sediments of cultured bacterial cultures, direct application of dissolved powder and sprayed supernatant. According to the results, the bacteria at the end of the logarithmic phase were tested with two batches of frozen strain. Instead of using the precipitate, the two strains were inactivated by heat and the inactivated bacteria were compared to the activated ones.

Measurement of cytokines; MMP-9 and PGE2 in primary human monocytes

[0052] Human primary monocytes were isolated from the buffy coat (concentrated suspension of leukocytes) of healthy human blood donors. Cells were seeded in 24-well plates for ELISA experiments. Cells were incubated with LPS for 24 hours. Probiotics (5 doses) were added 30 min before LPS treatment. After 24 hours, the pellet was removed, centrifuged, and assayed for IL-1beta, TNFalpha, IL-6, IL-8, MMP-9, isoprostane-8, and PGE2 concentrations in EIA assays (PGE2, from AssayDesign, isoprostane, from Cayman) or ELISA assays (all cytokines, Immunotools, MMP-9, GE Healthcare) using the manufacturer's protocol. Each dose was tested 2-3 times on two buffy coats from different donors.

[0053] First, different types of probiotic lyophilized powder preparations were tested for the stimulation of human monocytes.

[0054] Probiotics were collected and then centrifuged. Cells were dissolved in fresh medium and applied to human monocytes.

[0055] Monocytes were then incubated with probiotics for 30 minutes, and then LPS was added, and after 24 hours the precipitate was removed and used to determine inflammatory parameters.

Example 3: Testing of heat-inactivated strains

Establishing heat inactivation

[0056] Heat inactivation was established for two layers. At the end of the logarithmic phase of bacterial growth, an aliquot of the bacterial suspension was removed, added to a fresh 50 ml test tube and incubated for 5 min at 80°C in a water bath. 5 min at 80°C inactivated the bacteria and stopped their growth.

[0057] Testing of heat-inactivated strains revealed that the enhancing effects on IL-1 and TNF were not influenced by heat treatment. Furthermore, heat activation did not, or only slightly, affect PGE2 inhibition by both strains.

Example 4: Screening of probiotics on human monocytes and effect on NF-kappaB activation [0058] Various probiotic strains were screened in their activated and attenuated (heat-inactivated) form on LPS-induced human primary monocytes (determination of IL-1beta, IL-6, IL-8, TNFalpha, PGE2, 8-isoprostane and MMP-9.

[0059] A typical anti-inflammatory pattern of the new strain according to examples 1-4 is given in Figure 1.

[0060] Experiments testing the efficacy of TNF-induced NF-kappaB activation in NIH-3T3 fibroblasts were performed on a cell line containing a stably transfected luciferase gene driven by an NF-kappaB dependent promoter. Cells were stimulated with TNF in the presence or absence of probiotics. After 6 h of stimulation, the cells were lysed, and the luciferase activity was measured in a luminometer.

Example 5: Screening of selected probiotics on human gingival fibroblasts

[0061] The selected strain of the invention was applied to human gingival fibroblasts. Fibroblast cultures were maintained as described in the manufacturer's protocol. Before stimulation, cells were seeded in 24-well plates for ELISA experiments. Cells were incubated without (unstimulated control) or with IL-1beta for 24 h. Probiotics (5 doses, depending on the outcome of screening tests) were added 30 min before IL-1 treatment. After 24 h, the pellet was removed, centrifuged and assayed for IL-6, IL-8, isoprostane and PGE2 concentrations by EIA (PGE2, from AssayDesign, isoprostane from Cayman) or ELISA (IL-6, IL-8, Immunotools), using the manufacturer's protocol. Each dose was tested at least 2-3 times. The strains showed some IL-6 inhibitory effects.

[0062] The anti-inflammatory template strain according to Example 5 is shown in Figure 2.

Example 6: Probiotic lozenges or compresses

Production process:

[0064]

• components 1 and 6 are dried in a dryer with a vacuum section at 50 °C and under a pressure of max. 10 mbar for 16 hours

• all components are accurately measured

Components 1, 2, 3, 4 and 5 are combined and thoroughly mixed (block A). The probiotic material is administered in lyophilized form having an activity of about 105 to 1012 colony forming units (CFU) per gram.

• block A is additionally added to component 6 and thoroughly mixed for 5 minutes

• the powder mixture is then pressed into tablets in a tablet press EK0 (Korsch AG, Berlin) under a set pressure of 15 - 20 kN

Target parameters:

o tablet diameter: 20 mm

o tablet weight: 2.0 g

• storage at ST in closed aluminum bags. On 5 lozenges, 1 g of desiccant was used for dehumidification (activated by storage for 3 hours at 105°C in a dryer with a vacuum compartment). Example 7: Toothpaste powder

[0065]

1

Production process:

[0066]

• component 7 is dried in a dryer with a vacuum section at 50 °C and under a pressure of max. 10 mbar for 16 hours

• all components are accurately measured

• components 1,2,3 and 4 are combined and thoroughly mixed (block A)

• components 5 and 6 are, if necessary, combined and mixed with each other (block B). Probiotic material is administered in lyophilized form having an activity of 105 to 1012 colony forming units (CFU) per gram.

• blocks A and B are subsequently combined and thoroughly mixed

• the mixture is added to component 7 and thoroughly mixed for 5 minutes

• the powder mixture is divided into portions of 0.5 g per storage at ST with 1 g of desiccant per portion (activated by storage for 3 h at 105°C in a dryer with a vacuum compartment in closed aluminum bags.

Example 8: Toothpaste powder

[0067]

Production process:

[0068]

• components 6, 9 and 10 are dried in a dryer with a vacuum section at 50 °C under a pressure of max.

10 mbar 16 hours

• all components are accurately measured

• components 1,2,3,4,5 and 6 are combined and thoroughly mixed with each other (block A)

• components 7 and 8 are thoroughly mixed (block B). Probiotic material is administered in lyophilized form having an activity of about 10<5> to 10<12> colony forming units (CFU) per gram.

• blocks A and B are subsequently combined and thoroughly mixed with each other

• components 9 and 10 are combined and thoroughly mixed (block C)

• the two mixtures (blocks A/B and block C) are combined and thoroughly mixed for 5 minutes

• the powder mixture is pressed into tablets in a tablet press EK0 (Korsch AG, Berlin) at a set pressure of 15

- 20 kN

Target parameters:

o tablet diameter: 9 mm

o tablet weight: 0.3 g

• storage at ST in closed aluminum bags. 1 g of desiccant for dehumidification was used on 3 tablets (activated by storage for 3 hours at 105°C in a dryer with a vacuum compartment). Example 9: Chewing gum

[0069]

[0070]

• component 2 is dried in a dryer with a vacuum section at 50 °C and under a pressure of max. 10 mbar for 16 hours

• all components are accurately measured

• component 1 is tempered to 45 - 59 °C in a laboratory chewing gum mixer by mixing with heating until a homogeneous mass is obtained. The heating is on during the entire mixing process.

• components 2, 3 and 4 are subsequently added and mixed until the mixture becomes homogeneous and when the powder is no longer visible

• According to the formula, component 6 is inserted into component 5 (block C) or component 7 (block D). Probiotic material is administered in lyophilized form having an activity of approximately 10<5> to 10<12> colony forming units (CFU) per gram. The components are mixed until a uniform suspension is obtained.

• First, block C is added to the mass of chewing gum and mixed again until a homogeneous mass is obtained. • Finally, block D is processed accordingly. After adding the composition, it must be kneaded until a uniform mass of chewing gum is obtained.

• the mass is then removed from the mixer and mini-sticks are formed using an embossing roller using a 'slab' embossing set

• storage at ST in closed aluminum bags. 1 g of desiccant was used for dehumidification on 7 chewing gums (activated by storing for 3 hours at 105°C in a dryer with a vacuum compartment).

Example 10: probiotic balls

[0071]

1

Production process:

[0072] Production of calcium chloride baths for the deposition of alginate beads:

• 2% calcium chloride solution is made from distilled water and calcium chloride. Care must be taken to ensure that the CaCl2 is completely dissolved.

[0073] Production of alginate solution (pectin or gellan gum can be used instead of alginate): • in a reaction vessel with a mixer, which is adapted to the size of the batch, water is provided

• the mixer is turned on and while mixing on high, certain amounts of alginate, gum arabic, wheat fiber and probiotics are added, as well as optionally necessary gellan gum

• the mixture is heated to 80 °C during mixing and kept at that temperature for 5 minutes - during this step the gel-forming components dissolve

• after that, the heating is turned off and the solution with the warm gel is stirred for at least 30 minutes until there are no more lumps.

• after that, the solution is cooled in the refrigerator to 39 - 43 °C during mixing.

• in another vessel, flavor and color are added, if necessary, and thoroughly mixed, if flavor is not used, color is mixed with glycerol

• when the dye dispersion is homogeneously mixed, it is added to the batch vessel with the alginate solution. The mixing vessel is rinsed several times with approximately 10% of the alginate solution using water and added to the dispersion

• the alginate solution is further mixed for at least 5 minutes

• The batch is then further mixed for at least 15 minutes at low speed to remove any air that may be present.

Drips of alginate solution into calcium chloride solution for bead precipitation

[0074]

• the alginate dispersion is transferred to a tightly sealed pressure-stable reaction vessel that has two outlets. At one outlet, pressurized air is applied. Another outlet through the pipe leads to the nozzles of the drip unit.

• the reaction vessel is tempered over a hot plate so that the alginate solution reaches a temperature of approximately 45 °C. The solution is gently stirred with a magnetic stirrer.

• after applying pressure to the reaction vessel, the alginate solution is pressed towards the nozzles, which are set to oscillate by means of an oscillator. By adapting the pressure and frequency of the oscillator, the size of the resulting droplets at the tips of the nozzles can be adjusted.

• The drops of alginate solution formed at the tips of the nozzles fall into the collection vessel in the form of a funnel in which the calcium chloride solution, which has been prepared in advance, circulates.

• the hardened alginate beads together with the calcium chloride solution pass through the funnel and are collected in a sieve, the collected calcium chloride solution is pumped back into the funnel below the drip unit and recycled.

The balls are dried in an Aeromatic moving bed dryer at an inlet air temperature of 80 °C until an outlet air temperature of 45 °C is reached

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Claims (15)

PATENT REQUESTS 1. An isolated microorganism preparation containing or consisting of Lactobacillus plantarum GOS 42 (DSM 32131). 2. A composition containing the microorganism Lactobacillus plantarum GOS 42 (DSM 32131) together with a carrier, excipient and/or diluent. 3. The isolated preparation according to claim 1 or the composition according to claim 2, wherein the microorganism is present as vegetative cells and/or spores. 4. An isolated preparation according to claim 1 or 3 or a composition according to claim 2 or 3, wherein the microorganism is attenuated or dead. 5. The composition according to any one of claims 2-4, wherein the composition is a pharmaceutical composition. 6. The composition according to any one of claims 2-5, wherein the total amount of microorganisms is in the range of 0.01 to 100%, preferably in the range of 0.1 to 50%, more preferably in the range of 1 to 10%, in each case relative to the total weight of the composition, and/or wherein the total amount of microorganisms is in the range of 1 x 10<3> to 1 x 10<11> colony forming units (CFU), preferably in the range of 1 x 10<5> to 1 x 10<10>CFU. 7. The composition according to claim 6, wherein the total amount of microorganisms is in the range of 0.1 to 50% in relation to the total weight of the composition; and/or the total amount of microorganisms is in the range of 1 x 10<5> to 1 x 10<10>CFU. 8. Composition according to claim 7, wherein the total amount of microorganisms is in the range of 1 to 10% in relation to the total weight of the composition; and/or wherein the total amount of microorganisms is in the range of 1 x 10<8> to 1 x 10<9>CFU. 9. The composition according to any one of claims 2-8, wherein the composition is coated or encapsulated. 10. The composition according to any one of claims 2-9, wherein the composition is in the form of a solution, suspension, emulsion, tablet, granule, powder or capsule. 11. The composition according to any one of claims 2-10, wherein the composition is selected from the group consisting of toothpaste, dental gel, tooth powder, dentifrice, dentifrice, mouthwash, mouth spray, dental floss, chewing gum and lozenges. 12. A composition according to any one of claims 2-11, wherein the composition contains a food and/or beverage component for animals. 13. Isolated preparation or composition according to one of the previous requirements for medical use. 14. An isolated preparation or composition for use according to claim 13, wherein the medical use is for the treatment and/or prevention of inflammation. 15. An isolated preparation or composition for use according to claim 13 or 14, wherein the use in medicine is for reducing and/or inhibiting the release of one or more inflammatory factors selected from the group that includes interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), isoprostanes, matrix metallopeptidase 9 (MMP9) and NF-kB. 1